Novel therapeutic agent for a lung disease and/or method for screening for the same

ABSTRACT

Provided are a therapeutic and/or prophylactic agent for a lung disease and a method for screening for the therapeutic and/or prophylactic agent. Provided are a therapeutic and/or prophylactic agent for a lung disease comprising an Arid5A inhibitor as an active ingredient and a method for screening for the therapeutic and/or prophylactic agent.

TECHNICAL FIELD

The present invention relates to a novel therapeutic agent for a lungdisease and/or a method for screening for the novel therapeutic agent.

BACKGROUND ART

Lung diseases include many known inflammatory lung diseases such asacute bronchitis due to acute inflammation caused by viruses, bacteria,or the like; chronic bronchial asthma mainly developed from respiratorytract inflammation due to eosinophils; interstitial pneumonia involvinglung structure damage caused by alveolar epithelial inflammation; andchronic obstructive pulmonary disease (COPD), which is known as adisease characterized by chronic pulmonary inflammation caused byvarious factors, especially smoking, leading to alveolar walldestruction and bronchial mucous gland hypertrophy and the likeresulting in shortness of breath, increased cough and sputum, etc.

In the pathological process of the inflammatory lung diseases asdescribed above, it has been known that transmitters, called chemotacticfactors, released at early stage will stimulate inflammatory cells, suchas neutrophils, basophils, eosinophils, and macrophages and the like, tomigrate to local sites and the migrated inflammatory cells will releaseinjurious enzymes and radicals to damage tissues and at the same timewill release cytokine or the like, resulting in further migration andactivation of the inflammatory cells. When such inflammation develops inrespiratory tracts, the infiltrating inflammatory cells will damagebronchial and pulmonary tissues. This finally leads to impairment ofrespiratory function, including reduction in respiratory flow and oxygenexchange capacity, characteristic of each of the diseases. Lungstructure and function are further markedly suppressed, and many casesmay progress to intractable diseases that will eventually lead tofibrogenesis or honeycomb lung.

Such inflammatory lung diseases have been treated with drugs withanti-inflammatory effects, and mild to moderate bronchial asthma hasbeen known to completely response to adrenocortical steroids (see NonPatent Literature 1). Interstitial pneumonia has been also treated withadrenocortical steroids at acute exacerbation and combined withimmunosuppressive agents where appropriate. Further progression willcertainly induce hypoxemia and therefore require oxygen therapyincluding home oxygen therapy. Adrenocortical steroids have beenreported to prevent exacerbation of COPD but their effects on thepathology of COPD are limited (see Non Patent Literature 2). Pulmonaryfibrosis has been also treated with steroid therapy although lesionsoften remain and acute exacerbations are frequently observed due to sideeffects or reduction or withdrawal of steroidal agents. This is neversatisfying in clinical cure and therefore there are needs to developnovel therapeutic agents.

AT Rich Interactive Domain 5A (hereinafter abbreviated as Arid5A) hasbeen reported to be a protein that stabilizes IL-6 mRNA (see, e.g., NonPatent Literature 3).

CITATION LIST Non Patent Literature

-   Non Patent Literature 1: GINA Guideline, 2006-   Non Patent Literature 2: N. Engl. J. Med., 1999, 340 (25), 1948-1953-   Non Patent Literature 3: PNAS, 110, 23, 9404-9414 (2013)

SUMMARY OF INVENTION Technical Problem

However, the relationship between Arid5A and a lung disease has beenunknown.

The present invention intends to provide a more effective therapeuticagent for a lung disease and a method for screening for the therapeuticagent.

Solution to Problem

The present invention specifically includes the following:

-   [1] A therapeutic agent and/or prophylactic agent for a lung    disease, comprising an Arid5A inhibitor as an active ingredient.-   [2] The therapeutic agent and/or prophylactic agent for a lung    disease according to [1], wherein the Arid5A inhibitor is at least    one substance selected from the group consisting of a nucleic acid    oligo and an Arid5A antibody.-   [3] The therapeutic agent and/or prophylactic agent for a lung    disease according to [2], wherein the nucleic acid oligo is an oligo    consisting of a natural or non-natural RNA or DNA.-   [4] The therapeutic and/or prophylactic agent for a lung disease    according to any of [1] to [3], wherein the lung disease is    pulmonary fibrosis.-   [5] A method for screening for a candidate substance useful for the    treatment and/or prevention of a lung disease, wherein the method    comprises:    -   (a) detecting an effect of test agents on the expression of        Arid5A and    -   (b) selecting the agents that decrease the expression of Arid5A        as compared to the absence of the test agents.-   [6] A method for screening for a candidate substance useful for the    treatment and/or prevention of a lung disease, wherein the method    comprises:    -   (a) administering test agents to experimental animals,    -   (b) determining an effect on the expression of Arid5A by PCR,        and    -   (c) selecting the agents that decrease the expression of Arid5A        as compared to no administration of the test agents to the        experimental animals.-   [7] A method for screening for a candidate substance useful for the    treatment and/or prevention of a lung disease, wherein the method    comprises:    -   (a) detecting an effect of test agents on the function of Arid5A        and    -   (b) selecting the agents that decrease the function of Arid5A as        compared to the absence of the test agents.-   [8] The method for screening for a candidate substance useful for    the treatment and/or prevention of a lung disease according to [7],    wherein the function of Arid5A is to stabilize IL-6 mRNA.-   [9] The method for screening according to any of [5] to [8], wherein    the lung disease is pulmonary fibrosis.-   [10] A method for the treatment and/or prevention of a lung disease,    comprising administering an Arid5A inhibitor.-   [11] An Arid5A inhibitor for use in the treatment and/or prevention    of a lung disease.-   [12] Use of an Arid5A inhibitor for production of a therapeutic    and/or prophylactic agent for a lung disease.

Advantageous Effects of Invention

The therapeutic agent for a lung disease according to the presentinvention has high therapeutic efficacy for a lung disease. The presentinvention also provides a method for screening for a therapeutic agentwith high therapeutic efficacy.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A and 1B show the generation of Arid5A-deficient mice: FIG. 1A,top: the wild type allele of Arid5A, middle: the targeting vector, andbottom: the expected mutated allele. The main part of DNA binding domainis substituted with Neo-cassette interrupted by loxp sequences at bothends of the Neo-cassette. FIG. 1B, genotyping by Southern blottinganalysis; wild type (+/+), heterozygote (+/−).

FIG. 2 is a graph showing % survival of Arid5A-deficient mice and wildtype mice after inducing lung disorders by intratracheal instillation ofbleomycin.

FIGS. 3A and 3B present photographs of lung from an Arid5A-deficientmouse (FIG. 3A) and a wild type mouse (FIG. 3B) at day 7 afterintratracheal instillation of bleomycin.

FIGS. 4A and 4B present hematoxylin-eosin stained images of lung tissuesections from an Arid5A-deficient mouse (FIG. 4A) and a wild type mouse(FIG. 4B) at day 7 after intratracheal instillation of bleomycin.

FIG. 5 shows the amounts of IL6, TNF, and IFN-γ in lung lavage fluidsfrom Arid5A-deficient mice and wild type mice at day 7 afterintratracheal instillation of bleomycin.

FIG. 6 is a graph showing the levels of Arid5A mRNA quantified by qPCRin a macrophage cell line.

FIG. 7 is a graph showing the concentrations of IL-6 measured by ELISAin culture supernatants of a macrophage cell line.

FIG. 8 is a graph showing the concentrations of TNF-α measured by ELISAin culture supernatants of a macrophage cell line.

FIG. 9 is a graph showing mRNA expression of Arid5A in peritonealmacrophages.

FIG. 10 is a graph showing mRNA expression of IL-6 in peritonealmacrophages.

FIG. 11 is a graph showing mRNA expression of TNF-α in peritonealmacrophages.

DESCRIPTION OF EMBODIMENTS

The present invention will be now described in more detail below.

The present invention relates to a therapeutic agent for a lung diseaseand a method for screening for the therapeutic agent.

“Arid5A” in the present invention refers to AT Rich Interactive Domain5A (hereinafter abbreviated as Arid5A) and has been reported to be aprotein that stabilizes IL-6 mRNA. For example, human Arid5A includesvarious isoforms (NCBI accession numbers NP997646.1, XP006712266.1,XP006712265.1, XP005263918.1, XP005263913.1, XP005263917.1, etc.).Similarly, mouse Arid5A also includes various isoforms (NCBI accessionnumbers NP00165676.1, NP00165677.1, NP001277655.1, NP001277656.1,NP666108.2, etc.).

“Arid5A inhibitor” in the present invention refers to a substance thatinhibits the expression and/or function of Arid5A. The Arid5A inhibitormay be a substance that directly inhibits the expression itself ofArid5A or may be a substance that indirectly inhibits a biologicalfunction of Arid5A by binding to a molecule affected by Arid5A (e.g.,IL-6 mRNA). The Arid5A inhibitor preferably includes a substance thatinhibits stabilization of IL-6 mRNA by binding competitively with Arid5Ato IL-6 mRNA. For example, human IL-6 mRNA includes various isoforms(NCBI accession numbers NM000600.3, XM005249745.2, etc.). Similarly,mouse IL-6 mRNA also includes various isoforms (NCBI accession numberNM031168.1, etc.).

The Arid5A inhibitor according to the present invention includes, but isnot particularly limited to, for example, low-molecular substances suchas an anti-Arid5A antibody, a nucleic acid oligo, and chlorpromazine.Preferred examples of the Arid5A inhibitor according to the presentinvention include an siRNA.

Arid5A expression can be inhibited, for example, by utilizing RNAinterference effect on the expression of Arid5A gene. RNA interferenceis a method that has been reported to suppress gene expression using RNA(Genes and Development, 16, 948-958 (2002)) and is a phenomenon in whichthe expression of both a transduced foreign gene and an endogenoustarget gene is suppressed by transducing a duplex RNA having a sequenceidentical or similar to the sequence of target gene into cells.Specifically, an siRNA or an antisense nucleic acid having an RNAinterference effect on the expression of Arid5A gene can be used toinhibit the expression of Arid5A gene.

“Nucleic acid oligo” according to the present invention means a nucleicacid oligomer that controls the function of Arid5A gene or protein andincludes an siRNA, an shRNA, an antisense nucleic acid, a decoy nucleicacid, and a nucleic acid aptamer.

The nucleic acid oligo according to the present invention can includepreferably an siRNA or an shRNA. The siRNA means a duplex RNA consistingof a short strand having a length sufficient to avoid toxicity in cellsand can have, for example, 15 to 49 base pairs, suitably 15 to 35 basepairs, and more suitably 21 to 30 base pairs. The shRNA is another typeof RNA in which a single-stranded RNA forms a duplex by adopting ahairpin structure.

The siRNA and shRNA are not required to be completely identical to thetarget gene, but have a sequence homology of at least 70% or more,preferably 80% or more, more preferably 90% or more, and most preferably95% or more.

A duplex region, in which base pairing occurs, in an siRNA and shRNA mayinclude not only a region that has perfect pairing of bases but also aregion that has mispairing of bases due to mismatch (in whichcorresponding bases are not complementary), bulge (in which eitherstrand has no corresponding base), and the like. In the presentinvention, a region of duplex RNA, in which base pairing occurs, in adsRNA may comprise both bulge and mismatch.

“Antisense nucleic acid” in the present invention is an antisensenucleic acid complementary to the product transcribed from DNA encodingArid5A. Effects of the target gene expression suppressed by an antisensenucleic acid are caused by multiple factors as listed below. The factorsinclude degradation due to activity of an RNase that recognizes RNAduplex, inhibition of transcriptional initiation due to triplexformation, suppression of transcription due to hybridization with a sitehaving an open-loop structure locally formed by RNA polymerase,inhibition of transcription due to hybridization with RNA that is in theprocess of being synthesized, suppression of splicing due tohybridization at the junction between the intron and exon, suppressionof splicing due to hybridization with a spliceosome-forming site,suppression of translocation from nucleus to cytoplasm due tohybridization with mRNA, suppression of translation due to hybridizationwith a translation initiation factor binding site, blocking of peptidechain elongation due to hybridization with a coding region or polysomebinding site of mRNA, and suppression of gene expression due tohybridization with a site of interaction between nucleic acid andprotein. The expression of target gene can be suppressed by inhibitingthe process of transcription, splicing, or translation among others.

The antisense sequences used in the present invention may suppress theexpression of target gene with any effect as described above. In oneaspect, design of an antisense sequence complementary to a noncodingregion near 5′ end of Arid5A mRNA should be effective in the inhibitionof gene translation. However, sequences complementary to a coding regionor the 3′ noncoding region may be also used. Thus, sequencescomplementary to not only a coding region but also a noncoding region ofgene can be used. Hence, the antisense nucleic acids used in the presentinvention also include nucleic acids comprising antisense sequencesagainst not only coding regions but also noncoding regions of the gene.The antisense nucleic acids have complementarity to the transcriptionalproducts of the target gene of preferably 90% or more and mostpreferably 95% or more. The length of antisense RNAs that effectivelyinhibit the expression of target gene using the antisense sequences isnot particularly limited.

“Decoy nucleic acid” in the present invention is a nucleic acid oligohaving homology to the sequence of IL-6 mRNA recognized by Arid5A. Thedecoy nucleic acid binds Arid5A instead of the sequence of IL-6 mRNA andinhibits Arid5A function.

“Anti-Arid5A antibody” in the present invention can be obtained as apolyclonal antibody or a monoclonal antibody using any known means. Theantibody used in the present invention is derived from any source, whichis not particularly limited, and can include an antibody preferablyderived from mammals and more preferably derived from human. Monoclonalantibodies derived from mammals include an antibody produced by ahybridoma and an antibody produced by a host transformed with anexpression vector comprising an antibody gene using a geneticengineering technique. The antibodies inhibit the functional expressionof Arid5A by binding Arid5A.

Antibody-producing hybridomas can be produced essentially by using knowntechniques as described below. More specifically, Arid5A is used as asensitizing antigen for immunization according to a conventionalimmunization method. The resulting immune cells are fused with knownparent cells according to a conventional cell fusion method. The fusedcells are screened for a monoclonal antibody-producing cell according toa conventional screening method.

Specifically, an anti-Arid5A antibody may be produced as follows. Forexample, human Arid5A used as an antigen for obtaining the antibody canbe obtained using Arid5A gene/amino acid sequence disclosed in knownliteratures.

The sequence of Arid5A gene is inserted into any known expression vectorsystem. The resulting expression vector system is used to transformsuitable host cells. Arid5A protein of interest is then purified fromthe host cells or from the culture supernatant of the host cells usingany known method. The purified Arid5A protein may be used as asensitizing antigen. Arid5A protein produced by chemical synthesis maybe also used as a sensitizing antigen. A fusion protein between Arid5Aprotein and any other protein may be also used as a sensitizing antigen.

Mammals immunized with the sensitizing antigen are not particularlylimited, but are preferably selected in consideration of compatibilitywith parent cells used in cell fusion. Typically, rodents such as mouse,rat, and hamster are used.

Immunization of an animal with the sensitizing antigen is performedaccording to any known method. For example, the immunization isgenerally performed by intraperitoneally or subcutaneously injecting thesensitizing antigen into a mammal. Specifically, the sensitizing antigenis diluted with Phosphate-Buffered Saline (PBS), physiological saline,or the like to provide a suitable amount of suspension. The suspensionis optionally mixed with a conventional adjuvant, such as completeFreund's adjuvant, in a suitable amount and then emulsified. Theemulsified product is preferably administered to a mammal in some dosesevery 4 to 21 days. Suitable carriers can be also used in theimmunization of the sensitizing antigen.

After the immunization and confirmation of increase in the level of thedesired antibody in serum, immune cells are removed from the mammal andare submitted to cell fusion. Preferred immune cells submitted to cellfusion include particularly splenic cells.

The immune cells are fused with other parent cells. The other parentcells appropriately used include mammalian myeloma cells including avariety of known cell lines such as P3X63Ag8.653 (J. Immunol. 123,1548-1550 (1979)).

Cell fusion between the immune cells and myeloma cells can be performedessentially using any known method, for example, according to the methodof Milstein et al. (Methods Enzymol., 73, 3-46 (1981)).

More specifically, the cell fusion is performed, for example, in astandard nutrient medium in the presence of a cell fusion-promotingagent. The cell fusion-promoting agent that may be used includes, forexample, polyethylene glycol (PEG) and Sendai virus (HVJ). In addition,to increase fusion efficiency, auxiliary agents such as dimethylsulfoxide can be added and used as desired.

The immune cells and myeloma cells are preferably used, for example, asa ratio in which the number of immune cells is 1- to 10-times more thanthe number of myeloma cells. Culture media that can be used in the cellfusion include, for example, RPMI1640 medium and MEM medium, which bothare suitable for proliferation of the myeloma cell line, and otherconventional culture media used in this type of cell culture. Moreover,a serum complement such as fetal calf serum (FCS) can be also used.

In the cell fusion, a given amount of the immune cells and myeloma cellsare mixed thoroughly in the culture medium. Then, a PEG solutionpreheated to about 37° C., in which the PEG solution has, for example,an average molecular weight of about 1,000 to 6,000, is typically addedin a concentration of 30 to 60% (w/v) and mixed to form fusion cells ofinterest (hybridomas). Subsequently, to remove cell fusing agents andother agents unfavorable for the growth of the hybridomas, the followingsteps can be sequentially repeated: adding a suitable culture medium,centrifuging the resulting suspension, and removing the supernatant.

The hybridomas are selected by culturing the hybridomas in aconventional selective culture medium, such as HAT culture medium (aculture medium containing hypoxanthine, aminopterin, and thymidine). Theculture in HAT culture medium is continued for a period of timesufficient to kill any cells (non-fused cells) other than the hybridomasof interest, usually for a few days to weeks. The hybridomas producingthe antibody of interest are then screened for and cloned using aconventional limiting dilution method.

The desired human antibody having the binding activity to the desiredantigen or antigen-expressing cells can be obtained not only from thehybridomas by immunizing a nonhuman animal with the antigen but also bysensitizing human lymphocytes to the desired antigenic protein or thecells expressing the antigen in vitro and fusing the sensitized Blymphocytes with human myeloma cells, e.g., U266 (see Japanese PatentPublication No. 1-59878). Moreover, the desired human antibody may beobtained by administering the antigen or the cells expressing theantigen to a transgenic animal having human antibody gene repertoiresaccording to the method described above (see International PublicationNo. WO 93/12227, etc.).

The generated hybridomas producing a monoclonal antibody can besubcultured in a conventional culture medium and stored in liquidnitrogen over a long period of time.

A method for obtaining monoclonal antibodies from the hybridomasembraces a method of culturing the hybridomas according to anyconventional method and collecting the culture supernatant to obtain themonoclonal antibodies or a method of administering the hybridomas to amammal compatible with the hybridomas, allowing the hybridomas to beproliferated, and collecting the peritoneal fluid to obtain themonoclonal antibodies. The former method is suitable for obtaininghighly-pure antibodies while the latter method is suitable for highvolume production of antibodies.

The monoclonal antibodies used in the present invention may berecombinant antibodies, which are produced by a gene recombinationtechnique, which technique comprises cloning an antibody gene from ahybridoma, inserting the gene into a suitable vector, and transducingthe vector into a host (see, e.g., Borrebaeck C. A. K. and Larrick J. W.THERAPEUTIC MONOCLONAL ANTIBODIES, Published in the United Kingdom byMACMILLAN PUBLISHERS LTD, 1990).

Specifically, mRNA encoding the variable (V) region of the antibody ofinterest is isolated from cells producing the antibody, for example,hybridomas. The mRNA is isolated by preparing total RNA using any knownmethod, such as guanidine ultracentrifugation (Chirgwin, J. M. et al.,Biochemistry (1979) 18, 5294-5299) and the AGPC method (Anal. Biochem.(1987) 162, 156-159), to prepare mRNA, for example, using mRNAPurification Kit (from Pharmacia). Alternatively, mRNA can be directlyprepared by using QuickPrep mRNA Purification Kit (from Pharmacia).

The obtained mRNA is used with a reverse transcriptase to synthesizecDNA of the V region of antibody. The cDNA can be synthesized by usingAMV Reverse Transcriptase First-strand cDNA Synthesis Kit and the like.The cDNA can be also synthesized and amplified using 5′-RACE (Frohman,M. A. et al., Proc. Natl. Acad. Sci. USA (1988) 85, 8998-9002;Belyaysky, A. et. al., Nucleic Acid Res. (1989) 17, 2919-2932) with5′-Ampli FINDER RACE Kit (from Clontech) and PCR. The resulting PCRproducts are purified to obtain the DNA fragment of interest, and thefragment is linked to a vector DNA. Furthermore, the desired recombinantvector is prepared by using the linked vector DNA to make a recombinantvector, transducing the recombinant vector into Escherichia coli cellsor the like, and selecting colonies. The base sequence of the DNA ofinterest is confirmed using any known method, for example, the deoxymethod.

Once the DNA encoding the V region of the antibody of interest isobtained, this DNA is linked to a DNA encoding the desired constantregion (C region) of antibody and the linked DNA is inserted into anexpression vector. Alternatively, the DNA encoding the V region ofantibody may be inserted into an expression vector comprising the DNA ofthe C region of antibody.

For production of the antibody used in the present invention, anantibody gene is inserted into an expression vector to allow theantibody gene to be expressed under the control of an expressionregulatory region, such as enhancer and promoter, as described below.The expression vector is then transformed into host cells to permitexpression of the antibody.

Gene recombinant antibodies that have been artificially modified fordecreasing xenogeneic antigenicity against human and the like can beused in the present invention. The gene recombinant antibodies include,for example, chimeric antibodies, humanized antibodies, and humanantibodies. These modified antibodies can be produced using any knownmethod.

Chimeric antibodies are obtained by linking the DNA encoding the Vregion of antibody obtained as described above to the DNA encoding the Cregion of human antibody, inserting the linked DNA into an expressionvector, transducing the expression vector into a host, and allowing thehost to produce the chimeric antibodies (see European Patent ApplicationPublication No. EP125023, International Publication No. WO 92-19759).This known procedure can be used to obtain the chimeric antibodiesuseful for the present invention.

Humanized antibodies, also referred to as reshaped human antibodies orhuman-typed antibodies, are antibodies in whichcomplementarity-determining region (CDR) of antibody from a nonhumanmammal, such as mouse, is grafted into the complementarity-determiningregion of a human antibody. The general gene recombination techniquesfor producing humanized antibodies are known (see European PatentApplication Publication No. EP125023, International Publication No. WO92-19759).

Specifically, a DNA sequence that is designed to link CDRs of a mouseantibody to framework regions (FRs) of a human antibody is synthesizedusing PCR with some oligonucleotides produced to have overlappingsequences in the terminal parts of the oligonucleotides. The resultingDNA is linked to the DNA encoding the C region of a human antibody, andthen inserted into an expression vector. The expression vector istransduced into a host to result in production of the antibody (seeEuropean Patent Application Publication No. EP239400, InternationalPublication No. WO 92-19759).

The FRs of a human antibody linked to the CDRs are selected to allow thecomplementarity-determining regions to form a functional antigen bindingsite. One or more amino acids in the framework region of the variableregion of an antibody may be optionally substituted to allowcomplementarity-determining regions of a reshaped human antibody to forma functional antigen binding site as required (Sato, K. et al., CancerRes. (1993) 53, 851-856).

Chimeric antibodies and humanized antibodies have C regions of humanantibodies. The C regions of human antibodies include Cγ. For example,Cγ1, Cγ2, Cγ3, or Cγ4 can be used. The C regions of human antibodies maybe also modified for improving stability of the antibody or productionof the antibody.

Chimeric antibodies consist of variable regions of antibodies fromnonhuman mammals and C regions of human antibodies. Humanized antibodiesconsist of complementarity-determining regions of antibodies fromnonhuman mammals and framework regions and C regions of humanantibodies. Both chimeric antibodies and humanized antibodies have lowantigenicity in human bodies and therefore are useful for antibodiesused in the present invention.

The methods known to provide human antibodies include, in addition tothe methods as described previously, a technique of obtaining humanantibodies by the panning of a human antibody library. For example, avariable region of human antibodies can be expressed in the form ofsingle-chain antibody (scFv) on the surface of phages by phage display,and the phages that bind an antigen of interest can be selected. Theselected phages are analyzed for the genes by sequencing the DNAencoding variable regions of the human antibodies that bind the antigenof interest. Once the DNA sequences of scFvs that bind the antigen aredetermined, expression vectors suitable for the sequences can be made.The expression vectors can be used to obtain human antibodies. Thesemethods are well known and are described in WO 92/01047, WO 92/20791, WO93/06213, WO 93/11236, WO 93/19172, WO 95/01438, and WO 95/15388, whichcan be referenced.

The antibody gene constructed as described above can be expressed usingany known method, obtaining the antibody. For mammalian cells, theantibody can be expressed using a DNA that is obtained by operablylinking a useful promoter usually used, the antibody gene to beexpressed, and a poly A signal downstream of the 3′ end of the antibodygene, or a vector comprising the DNA. Promoters/enhancers can include,for example, human cytomegalovirus immediate early promoter/enhancer.

Other promoters/enhancers that can be used for antibody expression inthe present invention include a promoter/enhancer of viruses such asretrovirus, polyomavirus, adenovirus, and simian virus 40 (SV40), and apromoter enhancer from mammalian cells such as human elongation factor1α (HEF1α).

For example, the SV40 promoter/enhancer can be easily used according tothe method of Mulligan et al. (Mulligan, R. C. et al., Nature (1979)277, 108-114), and the HEF1α promoter/enhancer can be easily usedaccording to the method of Mizushima et al. (Mizushima, S. and Nagata,S. Nucleic Acids Res. (1990) 18, 5322).

For Escherichia coli, the antibody can be expressed by operably linkinga useful promoter usually used, a signal sequence for antibodysecretion, and the antibody gene to be expressed. The promoter caninclude, for example, lacZ promoter and araB promoter. The lacZ promotermay be used according to the method of Ward et al. (Ward, E. S. et al.,Nature (1989) 341, 544-546; Ward, E. S. et al., FASEB J. (1992) 6,2422-2427), and the araB promoter may be used according to the method ofBetter et al. (Better, M. et al., Science (1988) 240, 1041-1043).

The signal sequence for antibody secretion that is used for productionin the periplasm of Escherichia coli may be pelB signal sequence (Lei,S. P. et al., J. Bacteriol. (1987) 169, 4379-4383). The antibodyproduced in the periplasm is separated followed by appropriate refoldingof the structure of the antibody (see, e.g., WO 96/30394).

Origins of replication that can be used include those derived from SV40,polyomavirus, adenovirus, bovine papillomavirus (BPV), and the like.Moreover, the expression vectors can comprise, as a selection marker,aminoglycoside phosphotransferase (APH) gene, thymidine kinase (TK)gene, Escherichia coli xanthine-guanine phosphoribosyltransferase(Ecogpt) gene, dihydrofolate reductase (dhfr) gene, and the like, foramplifying the gene copy number in host cell systems.

Any production system may be used for producing the antibodies used inthe present invention. The production system for antibody productionincludes in vitro and in vivo production systems. The in vitroproduction system includes a production system using eukaryotic orprokaryotic cells.

The production system using eukaryotic cells includes a productionsystem using animal, plant, or fungal cells. The animal cells known tobe used include (1) mammalian cells, such as, CHO, COS, myeloma, babyhamster kidney (BHK), Hela, and Vero, (2) amphibian cells, such asXenopus oocyte, and (3) insect cells, such as sf9, sf21, and Tn5. Theplant cells known to be used include cells from Nicotiana tabacum, whichmay be used for callus culture. The fungal cells known to be usedinclude yeasts, such as genus Saccharomyces including Saccharomycescerevisiae, and filamentous bacteria, such as genus Aspergillusincluding Aspergillus niger.

The production system using prokaryotic cells includes a productionsystem using bacterial cells. The bacterial cells known to be usedinclude Escherichia coli (E. coli) and Bacillus subtilis.

These cells are transformed with an antibody gene of interest, and thetransformed cells can be cultured in vitro to obtain the antibody. Theculture is performed according to any known method. For example, culturemedia that can be used include DMEM, MEM, RPMI1640, and IMDM. A serumcomplement such as fetal calf serum (FCS) can be also used. The cellstransduced with the antibody gene may be also injected into peritonealcavity in animals to produce the antibody in vivo.

On the other hand, the in vivo production system includes a productionsystem using animals and plants. The production system using animalsincludes a production system using mammals or insects.

The mammals that can be used include goat, pig, sheep, mouse, and cattle(Vicki Glaserm, SPECTRUM Biotechnology Applications, 1993). The insectsthat can be used include silkworm. The plants that can be used include,for example, tobacco.

The antibody is produced in the animals or plants into which theantibody gene has been transduced, and collected. For example, theantibody gene is interrupted by a gene encoding a protein specificallyproduced in milk, such as goat βcasein, to prepare a fusion gene. TheDNA fragments comprising the fusion gene with the antibody gene aretransferred into goat embryos. The embryos are implanted into femalegoats. The desired antibody is obtained from milk produced by the goatsreceived the embryos, which are transgenic goats, or their offspring. Inorder to increase the amount of milk comprising the desired antibodyproduced by the transgenic goats, any suitable hormone may beadministered to the transgenic goats (Ebert, K. M. et al.,Bio/Technology (1994) 12, 699-702).

Silkworms are also used to obtain the desired antibody by infecting thesilkworms with a baculovirus having the antibody gene of interestinserted and collecting the fluid of the infected silkworms (Maeda, S.et al., Nature (1985) 315, 592-594). In addition, when tobacco is used,the antibody gene of interest is inserted into a plant expressionvector, such as pMON530, and the vector is transduced into a bacteriumsuch as Agrobacterium tumefaciens. The bacterium is used to infecttobacco such as Nicotiana tabacum, and the desired antibody is obtainedfrom leaves of the tobacco (Julian, K.-C. Ma et al., Eur. J. Immunol.(1994) 24, 131-138).

When an antibody is produced in the in vitro or in vivo productionsystem as described above, host cells may be co-transformed withexpression vectors into which a DNA encoding the heavy chain (H chain)or light chain (L chain) of the antibody is separately inserted or maybe transformed with a single expression vector into which DNAs encodingH chain and L chain are inserted (see International Publication No. WO94-11523).

The antibodies produced and expressed as described above can beseparated from intracellular or extracellular components or hosts andpurified to homogeneity. The antibodies used in the present inventioncan be separated and purified by affinity chromatography. Columns usedin affinity chromatography include, for example, protein A columns andprotein G columns. Carriers for protein A columns include, for example,Hyper D, POROS, and Sepharose F.F. Other details are not limited at allas long as the details are used for a method for separating andpurifying usual proteins.

For example, the antibody used in the present invention can be separatedand purified by appropriately selecting or combining a chromatographyexcept the affinity chromatography as described above, a filter,ultrafiltration, salt precipitation, dialysis, and the like. Thechromatography includes, for example, ion exchange chromatography,hydrophobic chromatography, and gel filtration. These chromatographiesare applicable to high performance liquid chromatography (HPLC). Reversephase HPLC may be also used.

The concentration of the antibody obtained above can be measured bydetermination of absorbance, ELISA, or the like. More specifically, inthe determination of absorbance, the antibody is suitably diluted withPBS (−) followed by determination of absorbance at 280 nm to calculatethe concentration of the antibody by setting the absorbance at theconcentration of 1 mg/ml to 1.350 D. In ELISA, the concentration of theantibody can be measured as follows. One hundred μl of goat anti-humanIgG (from TAG) diluted to 1 μg/ml with 0.1 M bicarbonate buffer (pH 9.6)is added to a 96-well plate (from Nunc) and incubated at 4° C. overnightto immobilize the antibody. After blocking, 100 μl of the antibody usedin the present invention or a sample containing the antibody that issuitably diluted, or human IgG (from CAPPEL) as a reference standard isadded and incubated at room temperature for an hour.

After washing, 100 μl of alkaline phosphatase-labelled human IgG diluted5,000-fold (from Bio Source) is added and incubated at room temperaturefor an hour. After washing, a substrate solution is added and incubated.The concentration of the antibody of interest is then calculated bydetermination of absorbance at 405 nm on the MICROPLATE READER Model3550 (from Bio-Rad).

The antibody used in the present invention may be an antibody conjugatedwith any of various molecules including polyethylene glycol (PEG), aradioactive substance, and a toxin. Such a conjugated antibody can beobtained by chemically modifying the antibody produced as describedabove. The methods of modifying an antibody have been established in theart. The “antibody” in the present invention includes also theconjugated antibodies.

The antibody according to the present invention includes not only adivalent antibody as typified by IgG, but also a monovalent antibody anda polyvalent antibody as typified by IgM as long as the antibody bindsArid5A. The polyvalent antibody according to the present inventionincludes a polyvalent antibody whose antigen binding sites are all thesame or a polyvalent antibody whose antigen binding sites are differentin part or in whole.

The antibody according to the present invention may be a bispecificantibody as long as the antibody binds Arid5A. The bispecific antibodyrefers to an antibody having variable regions that recognize differentepitopes, within a single antibody molecule. The epitopes may be presentin different molecules or in a single molecule. In other words, thebispecific antibody in the present invention can have antigen bindingsites that recognize different epitopes in Arid5A. The bispecificantibody can also have one recognition site recognizing Arid5A and theother recognition site recognizing an antigen except Arid5A.

The methods for producing bispecific antibodies are known. For example,a bispecific antibody can be produced by combining two antibodies thatrecognize different antigens. Each of the antibodies to be combined maybe a half molecule of an antibody having H chain or L chain or a quartermolecule of an antibody consisting of only H chain. Alternatively, afusion cell producing a bispecific antibody may be produced by fusinghybridomas that each produces different monoclonal antibodies.Furthermore, bispecific antibodies can be produced with geneticengineering techniques.

The antibody according to the present invention may be a low molecularweight antibody as long as the antibody binds Arid5A. The low molecularweight antibody includes an antibody fragment lacking a portion of awhole antibody, such as whole IgG. An antibody molecule may lack someportions as long as the antibody molecule binds Arid5A. The antibodyfragments in the present invention preferably comprise either a heavychain variable region (VH) or a light chain variable region (VL), orboth. The amino acid sequence of the VH or VL can have addition,deletion, and/or substitution. The antibody fragments may further lackeither VH or VL, or a part of both as long as the antibody fragmentsbinds Arid5A. The antibody fragments may be also chimerized orhumanized. Specific examples of the antibody fragment can include, forexample, Fab, Fab′, F(ab′)2, and Fv. Specific examples of the lowmolecular weight antibody can include, for example, Fab, Fab′, F(ab′)2,Fv, a single-chain Fv (scFv), Diabody, and a single-chain (Fv)2(sc(Fv)2). The antibody according to the present invention includesmultimers (e.g., dimer, trimer, tetramer, and polymer) of theseantibodies.

Antibody fragments can be produced by digesting an antibody with anenzyme. Enzymes known to produce antibody fragments include, forexample, papain, pepsin, and plasmin. Alternatively, antibody fragmentscan be produced by constructing DNAs encoding the antibody fragments,inserting the DNAs into expression vectors, and then allowing theexpression vectors to be expressed in suitable host cells (e.g., Co M.S. et al., J. Immunol. (1994) 152, 2968-2976, Better M. & Horwitz A. H.,Methods in Enzymology (1989) 178, 476-496, Pluckthun A. & Skerra A.,Methods in Enzymology (1989) 178, 497-515, Lamoyi E., Methods inEnzymology (1986) 121, 652-663, Rousseaux J. et al., Methods inEnzymology (1986) 121, 663-669, Bird R. E. & Walker B. W., TrendsBiotechnol. (1991) 9, 132-137).

Each of the enzymes for digestion cleaves an antibody at the specificposition to provide antibody fragments having specific structures asdescribed below. On the other hand, genetic engineering techniques canbe used to delete any portion of an antibody:

in papain digestion: Fab;in pepsin digestion: F(ab′)2 or F(ab′); andin plasmin digestion: Facb

An scFv is obtained by linking a VH and a VL of an antibody. In an scFv,the VH and VL are linked by a linker, preferably a peptide linker(Huston J. S. et al., Proc. Natl. Acad. Sci. USA (1988) 85, 5879-5883).The VH and VL in an scFv may be derived from any antibody describedherein. The peptide linker linking V regions is not particularlylimited. Any single-stranded peptide, for example, consisting of about 3to 25 residues, may be used for the linker.

The V regions can be linked by, for example, PCR as described above. Forthe linking of V regions by PCR, a DNA encoding a full or desiredpartial amino acid sequence encoded by a DNA sequence encoding an Hchain or the V region of an H chain of an antibody and a DNA sequenceencoding an L chain or the V region of an L chain of an antibody is usedas a template. Each of the DNAs encoding the V region of H chain and Lchain is amplified by PCR using primers having sequences correspondingto the sequences at both ends of the DNA to be amplified. A DNA encodinga peptide linker is then prepared. The DNA encoding the peptide linkercan be also synthesized by PCR. The primers used in this PCR have basesequences, which can connect to each of the amplified products of the Vregions separately synthesized, previously added to the 5′ end of theprimers. Next, a PCR reaction is performed using each of the DNAs of [VHDNA], [peptide linker DNA], and [VL DNA] with primers for assembly PCR.The primers for the assembly PCR are a combination of a primer that cananneal to the 5′ end of [VH DNA] and a primer that can anneal to the 3′end of [VL DNA]. In other words, the primers for the assembly PCRcomprise a set of primers that can be used to amplify a DNA encoding thefull sequence of the scFv to be synthesized. The [peptide linker DNA]has a base sequence previously added, which can connect to each of theDNAs of the V regions. The primers for the assembly PCR are used to linkthese DNAs, eventually producing the full-length scFv as an amplifiedproduct. Once the DNA encoding the scFv is produced, an expressionvector comprising the DNA and recombinant cells transformed with theexpression vector can be obtained using any conventional method. ThescFv can be also obtained by culturing the resulting recombinant cellsand allowing the cells to express the DNA encoding the scFv.

Diabody refers to a bivalent low molecular weight antibody constructedby gene fusion (Holliger P. et al., Proc. Natl. Acad. Sci. USA (1993)90, 6444-6448, EP 404097, WO 93/11161). Diabody is a dimer composed oftwo polypeptide chains. Generally, each of the polypeptide chainscomposing the dimer is linked by a linker between VL and VH in a singlechain. The linker for the polypeptide chains in the diabody typically istoo short to allow the VL and VH on the same chain to associate witheach other. Specifically, amino acid residues composing the linker haspreferably 2 to 12 residues, more preferably 3 to 10 residues, andparticularly about 5 residues. Therefore, the VL and VH encoded in asingle polypeptide chain are not able to form an scFv and therefore twoseparate polypeptide chains result in dimerization to form two Fvs.Consequently, the diabody has two antigen binding sites.

An sc(Fv)2 is a single-stranded low molecular weight antibody, in whichtwo VHs and two VLs are linked by linkers (Hudson P. J. & Kortt A. A.,J. Immunol. Methods (1999) 231, 177-189). An sc(Fv)2 can be produced,for example, by linking two scFvs by a linker. Alternatively, an sc(Fv)2can be also produced by linking two VHs and two VLs by linkers, startingfrom the N-terminus of the single-stranded polypeptide, in the order asdescribed below:

[VH]-[linker]-[VL]-[linker]-[VH]-[linker]-[VL].

It is noted that the order of two VHs and two VLs is not particularlylimited to the order as described above and any order is acceptable. Forexample, the orders as described below can be also included.

[VL]-[linker]-[VH]-[linker]-[VH]-[linker]-[VL]

[VH]-[linker]-[VL]-[linker]-[VL]-[linker]-[VH]

[VH]-[linker]-[VH]-[linker]-[VL]-[linker]-[VL]

[VL]-[linker]-[VL]-[linker]-[VH]-[linker]-[VH]

[VL]-[linker]-[VH]-[linker]-[VL]-[linker]-[VH]

A plurality of the linkers may be the same or different type.

The linkers that can be used to link variable regions of an antibodyinclude any peptide linker that can be incorporated by geneticengineering or a synthetic compound linker (e.g., the linker disclosedin Protein Engineering (1996) 9, 299-305). Peptide linkers are preferredin the present invention. The length of peptide linkers is notparticularly limited and can be appropriately selected by those skilledin the art according to any purpose. Generally, amino acid residuescomposing a peptide linker have 1 to 100 amino acids, preferably 3 to 50amino acids, more preferably 5 to 30 amino acids, particularlypreferably 12 to 18 amino acids (e.g., 15 amino acids). An amino acidsequence composing a peptide linker can be any sequence unless thesequence inhibits the binding effect of scFv.

Alternatively, synthetic compound linkers (chemical cross-linkers) canbe also used to link V regions. Cross-linkers that are commonly used forcross-linking of peptide compounds can be used in the present invention.The cross-linkers that can be used include, for example,N-hydroxysuccinimide (NHS), disuccinimidyl suberate (DSS),bis(sulfosuccinimidyl)suberate (BS3), dithiobis(succinimidyl propionate)(DSP), dithiobis(sulfosuccinimidyl propionate) (DTSSP), ethylene glycolbis(succinimidyl succinate) (EGS), ethylene glycol bis(sulfosuccinimidylsuccinate)(sulfo-EGS), disuccinimidyl tartrate (DST),disulfosuccinimidyl tartrate (sulfo-DST), bis[2-(succinimideoxycarbonyloxy)ethyl]sulfone (BSOCOES), and bis[2-(sulfosuccinimideoxycarbonyloxy)ethyl] sulfone (sulfo-BSOCOES).

The therapeutic or prophylactic agent for a lung disease in the presentinvention may include a pharmaceutically acceptable material such as apreservative or a stabilizer. The term pharmaceutically acceptable meansa pharmaceutically acceptable material that by itself may or may not betherapeutically effective for a lung disease and can be administered incombination with the therapeutic agent as described above. Thepharmaceutically acceptable material may also be a material that is nottherapeutically effective but has a synergistic or additive stabilizingeffect in combination with an Arid5A inhibitor.

The pharmaceutically acceptable materials include, for example, sterilewater, physiological saline, a stabilizer, an excipient, a bufferingagent, an antiseptic, a surfactant, a chelating agent (e.g., EDTA), anda bonding agent.

The surfactants in the present invention can include nonionicsurfactants typically including, for example, sorbitan fatty acid esterssuch as sorbitan monocaprylate, sorbitan monolaurate, and sorbitanmonopalmitate; and glycerol fatty acid esters having HLB 6 to 18 such asglyceryl monocaprylate, glyceryl monomyristate, and glycerylmonostearate.

The surfactants can also include anionic surfactants typicallyincluding, for example, alkyl sulfates, in which the alkyl group has 10to 18 carbon atoms, such as sodium cetyl sulfate, sodium lauryl sulfate,and sodium oleyl sulfate; polyoxyethylene alkyl ether sulfates, in whichthe average number of moles added of the ethylene oxide is from 2 to 4and the alkyl group has 10 to 18 carbon atoms, such as polyoxyethylenesodium lauryl sulfate; alkyl sulfosuccinate ester salts, in which thealkyl group has 8 to 18 carbon atoms, such as sodium laurylsulfosuccinate ester; natural surfactants, such as lecithin andglycerophospholipid; sphingophospholipids such as sphingomyelin; andsucrose fatty acid esters in which the fatty acid has 12 to 18 carbonatoms.

The agents according to the present invention can include one or morethan one surfactant as described above in combination. The preferredsurfactants used in the formulation according to the present inventionare polyoxyethylene sorbitan fatty acid esters, such as polysorbate 20,40, 60, or 80, and polysorbate 20 and 80 are particularly preferred.Polyoxyethylene-polyoxypropylene glycols as typified by poloxamer (e.g.,Pluronic F-68 (R)) are also preferred.

The buffering agent in the present invention can include phosphoricacid, citrate buffer, acetic acid, malic acid, tartaric acid, succinicacid, lactic acid, potassium phosphate, gluconic acid, caprylic acid,deoxycholic acid, salicylic acid, triethanolamine, fumaric acid, andother organic acids, or carbonate buffer, tris buffer, histidine buffer,and imidazole buffer.

Liquid formulations may be also prepared by dissolving the agentsaccording to the present invention in aqueous buffers known in the artof liquid formulation. The buffers have generally a concentration of 1to 500 mM, preferably 5 to 100 mM, and more preferably 10 to 20 mM.

The agents according to the present invention may include other lowmolecular weight polypeptides, proteins such as serum albumin, gelatin,and immunoglobulin, amino acids, saccharides such as polysaccharides andmonosaccharides, carbohydrates, and sugar alcohols.

In the present invention, saccharides, such as polysaccharide andmonosaccharide, and carbohydrates include, for example, dextran,glucose, fructose, lactose, xylose, mannose, maltose, sucrose,trehalose, and raffinose.

In the present invention, sugar alcohols can include, for example,mannitol, sorbitol, and inositol.

When used as an aqueous solution for injection, the agents according tothe present invention may be combined with, for example, physiologicalsaline, an isotonic solution containing glucose or other adjuvants e.g.,D-sorbitol, D-mannose, D-mannitol, and sodium chloride, a suitabledissolution aid, for example, alcohols (e.g., ethanol), polyalcohols(e.g., propylene glycol, PEG), and nonionic surfactants (e.g.,polysorbate 80, HCO-50).

The agents according to the present invention may further comprise adiluent, a dissolution aid, a pH adjusting agent, a soothing agent, asulfur-containing reducing agent, an antioxidant, and the like, asdesired.

If necessary, the agents according to the present invention may be alsoencapsulated in microcapsules (microcapsules made of hydroxymethylcellulose, gelatin, poly[methyl methacrylic acid], or the like) or maybe adapted for colloidal drug delivery systems (such as liposome,albumin microsphere, microemulsion, nanoparticle, and nanocapsule) (seee.g., “Remington's Pharmaceutical Science 16th edition”, Oslo Ed.,1980). Moreover, methods for converting an agent to a sustained-releaseagent are known and are applicable to the present invention (Langer etal., J. Biomed. Mater. Res. 1981, 15: 167-277; Langer, Chem. Tech. 1982,12: 98-105; U.S. Pat. No. 3,773,919; European patent applicationpublication No. (EP) 58,481; Sidman et al., Biopolymers 1983, 22:547-556; EP133,988).

Pharmaceutically acceptable carriers to be used are appropriatelyselected from, but not limited to, those as described above or acombination thereof depending on the dosage form.

The Arid5A inhibitor according to the present invention can be used as apharmaceutical for human or other animals not only by directlyadministering the substance to patients but also by administering aformulation formulated using any known pharmaceutical method. Forformulation, a pharmaceutically acceptable material as described abovemay be added.

All agents in the present invention can be administered orally,parenterally, systemically, or locally in the form of pharmaceuticalpreparation. The route of administration can be appropriately selectedfrom, for example, intravenous injection including infusion,intramuscular injection, intraperitoneal injection, subcutaneousinjection, a suppository, intestinal infusion, and an oralenteric-coated formulation, depending on age and condition of a patient.An effective dosage is selected from the range from 0.001 mg to 100 mgper kg of body weight per dose. Alternatively, a dosage can be selectedfrom the range from 0.1 to 1,000 mg, preferably 0.1 to 50 mg perpatient. For example, the effective dosage for anti-Arid5A antibody isan amount that results in free antibody present in blood, and specificexamples of the preferred dosage and route of administration include 0.1mg to 40 mg, preferably 1 mg to 20 mg per kg of body weight per month (4weeks) as a single dose or divided doses, for example, a schedule foradministration including twice a week, once a week, once every twoweeks, and once every four weeks, by intravenous injection includinginfusion, subcutaneous injection, or the like. The schedule foradministration can be adjusted, for example, by extending the intervalbetween doses from twice a week or once a week to once every two weeks,once every three weeks, or once every four weeks while the condition ofpatient and the values of blood tests following doses are observed.

The lung disease according to the present invention include a wide rangeof diseases affecting lung and respiratory tract, for example, acutebronchitis, bacterial pneumonia, lung abscess, pulmonary tuberculosis,atypical mycobacterial lung infection, fungal lung disease, parasiticlung disease, opportunistic infection (Pneumocystis pneumonia,cytomegalovirus pneumonitis), aspiration pneumonia, common coldsyndrome, infectious respiratory diseases such as influenza; airwayobstructive diseases such as chronic obstructive pulmonary disease(COPD) and diffuse panbronchiolitis; allergic lung diseases such asbronchial asthma, hypersensitivity pneumonitis, eosinophilicpneumonitis, allergic bronchopulmonary aspergillosis, drug-inducedpneumonia, and eosinophilic granulomatosis with polyangiitis;interstitial lung diseases such as idiopathic interstitial pneumonia,radiation pneumonitis, sarcoidosis, idiopathic organizing pneumonia, andlung disease related to collagen vascular disease; neoplastic lungdiseases such as lung cancer, metastatic lung tumor, pulmonary benigntumor, and mediastinal neoplasm; pulmonary vascular lesions such aspulmonary thromboembolism, pulmonary arterial pulmonary hypertension,and pulmonary edema; pleural diseases such as pleurisy, pyothorax,pleural tumors, and pneumothorax; and respiratory failure such as acuterespiratory distress syndrome (ARDS) and chronic respiratory distresssyndrome.

The therapeutic and/or prophylactic agent for a lung disease accordingto the present invention also provide(s) agents useful for the treatmentof pulmonary fibrosis that is not limited by causes of the lung diseasesas described above. Pulmonary fibrosis is a disease characterized bydiffuse fibroplasia of the alveolar walls and main symptoms of dry coughand exertional dyspnea. Pulmonary fibrosis refers to an end-stagedisease state of interstitial pneumonia in a narrow sense while it meansa co-existing state of pulmonary fibrosis and interstitial pneumonia ina broad sense.

The present invention relates to a method for screening for apharmaceutical composition for treating or preventing a lung disease bysuppressing the expression and/or function of Arid5A.

The method for screening according to the present invention firstcomprises detecting an effect of test agents on the expression and/orfunction of Arid5A. The function of Arid5A includes, for example,contribution to stabilization of IL-6 mRNA by specifically binding tothe stem-loop of IL-6 mRNA and antagonistically inhibiting the action ofRegnase-1, which specifically destroys IL-6 mRNA. Accordingly, theeffect on the expression and/or function of Arid5A include, but are notlimited to, for example, suppression of the expression of Arid5A,inhibition of the function of Arid5A by binding to Arid5A, andinhibition of the function of Arid5A by binding competitively withArid5A to IL-6 mRNA.

The screening method also comprises selecting the agents that decreasethe expression and/or function as compared to the absence of the testagents.

In the first aspect of the screening method according to the presentinvention, Arid5A is first contacted with test agents.

The amino acid sequence of human Arid5A used in the method according tothe present invention is as described above. The Arid5A used in themethod according to the present invention includes a proteinfunctionally equivalent to the known Arid5A as described above. Such aprotein includes, but is not limited to, for example, a mutant, allele,variant, and homolog of Arid5A, a partial peptide of Arid5A, or a fusionprotein with another protein.

The mutant of Arid5A in the present invention can include a protein thatis a naturally occurring protein consisting of an amino acid sequence inwhich one or more amino acids in the amino acid sequence as describedabove are substituted, deleted, inserted, and/or added and isfunctionally equivalent to the protein consisting of the amino acidsequence as described above. The mutant of Arid5A can also include aprotein that is encoded by a naturally occurring DNA hybridizing to aDNA consisting of the base sequence as described above under a stringentcondition and is functionally equivalent to a protein consisting of theamino acid sequence as described above.

In the present invention, the number of mutated amino acids is notparticularly limited but should be typically 30 amino acids or less,preferably 15 amino acid or less, more preferably 5 amino acids or less(for example, 3 amino acids or less). When mutated, the amino acidresidues are desirably mutated to other amino acids having preservedside chain properties. For example, the properties of amino acid sidechains can include hydrophobic amino acids (A, I, L, M, F, P, W, Y, V),hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), amino acidshaving aliphatic side chains (G, A, V, L, I, P), amino acids having sidechains containing hydroxy groups (S, T, Y), amino acids having sidechains containing sulfur atoms (C, M), amino acids having side chainscontaining carboxylic acids and amides (D, N, E, Q), amino acids havingside chains containing bases (R, K, H), and amino acids having aromaticside chains (H, F, Y, W) (each letter in parentheses representsone-letter notation for amino acids). Even though a polypeptide has anamino acid sequence modified with deletion and/or addition of one ormore amino acid residues, and/or substitution with other amino acidsequences for an amino acid sequence, the polypeptide is known tomaintain its biological activity.

“Functionally equivalent” in the present invention refers to a proteinof interest having a biological or biochemical function equivalent toArid5A. Arid5A contributes to stabilization of IL-6 mRNA by specificallybinding to the stem-loop of IL-6 mRNA and antagonistically inhibitingthe action of Regnase-1, which specifically destroys IL-6 mRNA. Thebiological or biochemical function of Arid5A in the present inventioncan include stabilization of IL-6 mRNA.

Methods well known to those skilled in the art for preparing a DNAencoding “a protein functionally equivalent to” a protein of interest,include methods using a hybridization technique and a polymerase chainreaction (PCR) technique. In other words, those skilled in the artgenerally could isolate a DNA that has high homology to Arid5A by usingthe base sequence of Arid5A or a part thereof as a probe and anoligonucleotide specifically hybridizing to Arid5A as a primer. The DNAin the present invention also includes a DNA encoding a protein having afunction equivalent to Arid5A that can be isolated by using suchhybridization technique and PCR technique.

The isolation of DNA is achieved by a hybridization reaction preferablyunder a stringent condition. The stringent hybridization condition inthe present invention refers to a condition in 6M urea, 0.4% SDS,0.5×SSC or a hybridization condition of stringency equivalent thereto. Acondition of higher stringency, for example, a condition in 6M urea,0.4% SDS, 0.1×SSC can be used to isolate a DNA having higher homology toArid5A. The DNA isolated under this condition should have high homologyto an amino acid sequence of the protein of interest in the amino acidlevel. The high homology refers to a sequence identity of at least 50%or more, more preferably 70% or more, and even preferably 90% or more(e.g., 95%, 96%, 97%, 98%, 99%, or more) throughout the amino acidsequence. The identity of amino acid sequences and base sequences can bedetermined using the algorithm BLAST by Karlin and Altschul (Proc. Natl.Acad. Sci., ISA 87: 2264-2268, 1990, Proc Natl Acad Sci USA 90: 5873,1993). Programs, called BLASTN and BLASTX, based on the algorithm ofBLAST have been developed (Altschul S F, et al: J Mol Biol 215: 403,1990). BLASTN is performed to analyze base sequences using parameters,for example, score=100, word length=12. BLASTX is performed to analyzeamino acid sequences using parameters, for example, score=50, wordlength=3. BLAST and Gapped BLAST programs are performed using thedefault parameters of each program. Specific procedures of theseanalyses are known.

The species of organisms from which Arid5A used in the method accordingto the present invention is derived is not limited to a particularspecies of organism. The species of organisms includes, for example,human, monkey, mouse, rat, guinea pig, pig, cattle, yeast, and insect.

The form of Arid5A used in the first aspect is not particularly limited.Arid5A may be, for example, in a purified form, in a form expressedwithin cells, and in a form expressed in cell extracts.

Arid5A can be purified using any well-known method. Cells expressingArid5A include cells expressing endogenous Arid5A or cells expressingforeign Arid5A. The cells expressing endogenous Arid5A include, but arenot limited to, cells from tissues of animals and cultured cells. Thecultured cells are not particularly limited and may be, for example,commercially-available cells. The species of organisms from which thecells expressing endogenous Arid5A is derived is not particularlylimited, but includes human, monkey, mouse, rat, guinea pig, pig,cattle, yeast, and insect. The cells expressing foreign Arid5A can beproduced, for example, by transducing a vector comprising a DNA encodingArid5A into cells. The transduction of the vector into cells can beperformed using any conventional method, such as, calcium phosphateprecipitation, electroporation, Lipofectamine method, andmicroinjection. The cells comprising foreign Arid5A can be alsoproduced, for example, by inserting a DNA encoding Arid5A into achromosome using a gene transfer technique utilizing homologousrecombination. The species of organisms from which the cells transducedwith foreign Arid5A are derived is not particularly limited to mammalsand may be any species of organisms that has an established technique tocause intracellular expression of a foreign protein.

Cell extracts containing the expressed Arid5A include, for example, acell extract containing an in vitro transcription-translation system anda vector comprising a DNA encoding Arid5A. The in vitrotranscription-translation system is not particularly limited and may beany commercially-available in vitro transcription-translation kit.

“Test agents” in the present invention are not particularly limited andcan include, for example, a single substance such as a natural compound,an organic compound, an inorganic compound, a nucleic acid, a protein,and a peptide, and an expression product from a compound library, anucleic acid library, a peptide library, or a gene library, a cellextract, a cell culture supernatant, a product from fermentationmicroorganisms, an extract of marine organisms, a plant extract, aprokaryotic cell extract, an extract of eukaryotic unicellularorganisms, and an animal cell extract. The test agents can beappropriately labeled and used as necessary. The labels include, forexample, a radioactive label and a fluorescent label. “Test agents” inthe present invention may include only one of the test agents as listedabove or may include a mixture of a plurality of the test agents.

“Contacting” in the present invention is performed depending on the formof Arid5A. For example, when Arid5A is in a purified form, thecontacting can be performed by adding a test agent to a purifiedpreparation. When Arid5A is in a form expressed within cells or in aform expressed in cell extracts, the contacting can be performed byadding a test agent to the cell culture medium or the cell extractrespectively, or by directly administering test agents to experimentalanimals. When the test agent is a protein, the contacting can be alsoperformed, for example, by transducing a vector comprising a DNAencoding the protein into cells expressing Arid5A or by adding thevector to an extract of cells expressing Arid5A. For example, thetwo-hybrid method using yeast or animal cells may also be used.

In the first aspect, the expression and/or function of Arid5A is thendetermined. The function of Arid5A can include stabilization of IL-6mRNA. Specifically, the function of Arid5A can be indirectly determined,for example, by contacting Arid5A with test agents and determining asuppressing effect on the degradation of IL-6 mRNA. Test agents thatdecrease or increase the expression and/or function as compared to theabsence of the test agents are then selected. When test agents areadministered to experimental animals, a tissue such as spleen is removedand the expression and/or function of Arid5A or the expression level ofIL-6 mRNA is determined in particular cells such as macrophage. Theexpression level can be determined using a technique appropriatelyselected from an amplification-based technique such as polymerase chainreaction (PCR), reverse transcription-polymerase chain reaction(RT-PCR), or real time PCR, a hybridization-based technique, and/orother detection techniques. “PCR” in the present application includestechniques determining various DNAs and RNAs by PCR andamplification-based techniques derived therefrom. It is known thatinhibition of Arid5A expression within cells will suppress theexpression of IL-6 mRNA and will have no effect on the expression ofTNF-α mRNA because Arid5A is a protein that contributes to stabilizationof IL-6 mRNA. The screening of test agents can be achieved by confirmingthat the test agents have effects on the expression of IL-6 mRNA and noeffect on the expression of TNF-α mRNA. Consequently, the test agentshaving an effect on Arid5A expression are selected, and thus the testagents having the effect of treating or preventing a lung disease areselected. The selected test agents include an agent that decreases theexpression and/or function of Arid5A and thus should have an effect oftreating or preventing a lung disease by suppressing the expressionand/or function of Arid5A.

In the second aspect, Arid5A is first contacted with a test agent andthen the binding of Arid5A to the test agent is detected. The detectionmethod is not particularly limited. The binding of Arid5A to the testagents can be detected, for example, by detecting a label (e.g., a labelthat can be quantitatively determined, such as a radioactive label and afluorescent label) attached to the test agents bound to Arid5A. Alabeling agent may be also attached to Arid5A. The binding can be alsodetected by immobilizing test agents or Arid5A onto a resin, chip, orthe like. The binding can be also detected based on the change of Arid5Afunction due to the binding of Arid5A to test agents.

In this aspect, test agents that bind to Arid5A are then selected. Theselected test agents include an agent that decreases the expression orfunction of Arid5A and thus the selected test agents should have theeffect of treating or preventing a lung disease by suppressing theexpression or function of Arid5A.

In the third aspect of the method for screening according to the presentinvention, cells expressing Arid5A are first contacted with a test agentand then the expression level of Arid5A is measured. The expressionlevel of Arid5A can be measured using any method known to those skilledin the art. The transcriptional level of the gene can be measured, forexample, by extracting mRNA of the gene according to any conventionalmethod and performing Northern hybridization or RT-PCR using the mRNA asa template. The expression level of the gene can be further measuredusing a DNA array technique.

The translation level of gene can be also measured by collecting afraction containing Arid5A encoded by the gene according to anyconventional method and detecting the expression of Arid5A usingelectrophoresis such as SDS-PAGE. The translation level of gene can bealso measured by performing Western blotting with an antibody directedagainst Arid5A to detect the expression of Arid5A. The antibody directedagainst Arid5A may be an antibody as described above.

In the third aspect, the test agents that decrease the expression levelof Arid5A as compared to the absence of the test agents are thenselected. The selected agents include an agent that decreases theexpression of Arid5A, and thus the selected agents should have theeffect of treating or preventing a lung disease by suppressing theexpression of Arid5A. It is known that inhibition of Arid5A expressionwithin cells will suppress the expression of IL-6 mRNA and will have noeffect on the expression of TNF-α mRNA because Arid5A is a protein thatcontributes to stabilization of IL-6 mRNA. The screening of test agentscan be achieved by confirming that the test agents have an effect on theexpression of IL-6 mRNA and no effect on the expression of TNF-α mRNA.Consequently, the test agents having an effect on Arid5A expression areselected, and thus the test agents having the effect of treating orpreventing a lung disease are selected.

In the fourth aspect of the screening method according to the presentinvention, provided are cells or cell extracts that have a DNA operablylinked to a reporter gene downstream of the promoter region of the DNAencoding Arid5A.

In the fourth aspect, “operably linked” refers to the linking betweenthe promoter region of Arid5A gene and a reporter gene to result in theexpression of the reporter gene induced by binding of a transcriptionfactor to the promoter region of Arid5A gene. Thus, the meaning of theterm “operably linked” includes an expression of a fusion proteininduced by binding a transcription factor to the promoter region ofArid5A gene even though a reporter gene is linked to another gene toform a fusion protein with another gene product.

The reporter gene is not particularly limited as long as its expressionis detectable.

The reporter gene includes, for example, CAT gene, lacZ gene, luciferasegene, β-glucuronidase gene (GUS), and GFP gene, which are commonly usedby those skilled in the art. The reporter gene also includes a DNAencoding Arid5A.

Cells or cell extracts that have a DNA operably linked to a reportergene downstream of the promoter region of the DNA encoding Arid5A can beprepared using the above-mentioned methods.

In the fourth aspect, the cells or cell extracts are then contacted withtest agents. The expression level of the reporter gene in the cells orcell extracts is measured.

The expression level of the reporter gene can be measured using anymethod known to those skilled in the art depending on the type of thereporter gene used. For example, when the reporter gene is CAT gene, theexpression level of the reporter gene can be measured by detectingchloramphenicol acetylated by the product of the gene. When the reportergene is lacZ gene, the expression level of the reporter gene can bemeasured by detecting the color reaction of the dye compound due tocatalysis of the expression product of the gene. When the reporter geneis luciferase gene, the expression level of the reporter gene can bemeasured by detecting fluorescence from the fluorescent compound due tocatalysis of the gene product. When the reporter gene is β-glucuronidasegene (GUS), the expression level of the reporter gene can be measured bydetecting luminescence of Glucuron (ICN) or the color reaction of5-bromo-4-chloro-3-indolyl-β-glucuronide (X-Gluc) due to catalysis theexpression product of the gene.

When Arid5A gene is used as a reporter, the expression level of the genecan be measured using any of the methods as described above.

In the fourth aspect, test agents are then selected that decrease orincrease the expression level of the reporter gene as compared to theabsence of the test agents. The selected test agents include an agentthat decreases the expression of Arid5A, and thus the selected testagents should have the effect of treating or preventing a lung diseaseby suppressing the expression of Arid5A.

In the fifth aspect, IL-6 mRNA is first contacted with a test agent andthen the binding of IL-6 mRNA to the test agent is detected. Thedetection method is not particularly limited. The binding of IL-6 mRNAto the test agents can be detected, for example, by detecting a label(e.g., a label that can be quantitatively determined, such as aradioactive label and a fluorescent label) attached to the test agentsbound to IL-6 mRNA. A labeling agent may be also attached to IL-6 mRNA.The binding can be also detected by immobilizing test agents or IL-6mRNA onto a resin, chip, or the like. The binding can be also detectedbased on the change of IL-6 mRNA activity due to the binding of IL-6mRNA to test agents.

In this aspect, test agents that bind to IL-6 mRNA are then selected.The selected test agents include an agent that decreases the expressionand/or function of Arid5A, and thus the selected test agents should havethe effect of treating or preventing a lung disease by suppressing theexpression or function of Arid5A.

In the sixth aspect, IL-6 mRNA is first contacted with a test agent andfurther contacted with Arid5A, and then the function of Arid5A isdetermined. The function of Arid5A can be determined in the manner asdescribed above. Test agents that decrease or increase the function ascompared to the absence of the test agents are then selected. Theselected test agents include an agent that decreases the function ofArid5A and thus should have an effect of treating or preventing a lungdisease by suppressing the function of Arid5A.

EXAMPLES Example 1. Generation of Arid5A Knockout Mice (FIGS. 1A and 1B)

C57BL/6 wild type mice (aged 6 to 8 weeks) were obtained from CLEAJapan, Inc. Arid5A knockout mice (No. CDB0602K:www.cdb.riken.jp/arg/mutant %20mice %20list.html) were generated usingthe method described at www.cdb.riken.jp/arg/Methods.html. The targetingvector was constructed by substituting the main part of DNA bindingdomain of Arid5A gene with Neo-cassette interrupted by lox P sequencesat both ends of the Neo-cassette and was transduced into ES cell strainTT2 by electroporation. Gene disruption by homologous recombination wasconfirmed by Southern blotting with a probe corresponding to the 3′ UTRof Arid5A gene. The resulting mutant ES cells were used to generatechimeric mice. Germline transmission of the mutant allele was confirmedby Southern blotting analysis and genomic PCR (primer 1:5′-ATACTTTCTCGGCAGGAGCA-3′; primer 2: 5′-TGAATGAACTGCAGGACG AG-3′) usinggenomic DNA from offspring mice obtained by crossing the chimeric micewith C57BL mice. The heterozygous mutant mice were raised in a specificpathogen-free environment and were back-crossed with C57BL mice for fivegenerations.

Example 2: Induction of Lung Disorders in Arid5A Knockout Mice (LungDisease Model)

Arid5A knockout mice (Arid5A−/−) and wild type mice aged 8 to 9 weeks(each mouse has a body weight of 20 to 25 g) intraperitoneally receivedtribromoethanol (Avertin) for anesthesia at 40 ng per mouse.Subsequently, bleomycin (BLM) was intratracheally instilled at a dose of2 mg/kg.

Survival Rate

The survival rate of the mice described above was examined for 4 weeks.

As a result, the survival rate of the wild type mice graduallydecreased, and all the wild type mice died after 3 weeks. In contrast,few Arid5A knockout mice developed lung disorders, and their survivalrate after 4 weeks was 80% (FIG. 2).

Observation of Lung Tissues

Three and seven days after intratracheal instillation of BLM, 10%neutral phosphate buffered formalin solution was intratracheallyadministered to the mice as described above and lung tissues wereisolated after fixation. Photographs of lung tissues from an Arid5Aknockout mouse and a wild type mouse 7 days after administration of BLMare shown in FIGS. 3A and 3B. Moreover, isolated lung tissues wereembedded in paraffin and sliced into 6 μm thick sections which werestained with hematoxylin and eosin (H&E) (FIGS. 4A and 4B).

As a result, the lung from the wild type mouse had inflammation andatrophy due to intratracheal instillation of BLM and accumulation ofneutrophils was observed (FIG. 3B, FIG. 4B) whereas the condition of thelung from the Arid5A knockout mouse remained the same as that of normallung (FIG. 3A, FIG. 4A).

Preparation and Analysis of Lung Lavage Fluids

Lung lavage fluids 7 days after intratracheal instillation of BLM (BALfluids) were collected in order to evaluate the accumulation ofinflammatory cells in pulmonary alveoli. Specifically, mice weretracheally intubated and instilled with 1.5 mL of sterile physiologicalsaline, and lavage fluids were then collected in two portions. Thisprocedure was performed a total of 3 times per mouse. The amount of lunglavage fluids collected was on average about 90% of the instilledamount. The collection rates of lavage fluids were substantiallyconstant regardless of treatment conditions or types of mice (wildtypeand Arid5A knockout mice). The collected lung lavage fluids werecentrifuged at 300×g for 10 minutes to collect cells which were countedwith a hemocytometer. The fraction of white blood cells was evaluatedbased on a cytological preparation. Specifically, slides were preparedusing Cytospin (TOMY SEIKO CO., LTD., Tokyo, Japan) and stained withDiff-Quik (from SYSMEX INTERNATIONAL REAGENTS CO., LTD.). The amounts ofIL-6, TNFα, and IFNγ in lung lavage fluids were also measured (FIG. 5).

As a result, as shown in FIG. 5, wild type mice had IL-6 concentrationof about 150 pg/mL, which were detected in lung lavage fluids 7 daysafter intratracheal instillation of BLM, while Arid5A knockout mice hadIL-6 concentration of about 30 pg/mL. No TNFα and IFNγ were detected inboth wild type mice and Arid5A knockout mice.

Example 3: Control of IL-6 expression controlled by Arid5A siRNA

The murine macrophage cell line RAW 264.7 was transduced with Arid5AsiRNA (SASI_Mm02_00341523 (sigma)) or a control siRNA (100 nM) byelectroporation. After culturing for 24 hours, the cells were stimulatedwith LPS (100 ng/mL) for 24 hours. RNA was extracted from the resultingcells. The level of Arid5A mRNA was quantified by qPCR (FIG. 6). Theconcentrations of IL-6 and TNF-α in each culture supernatant werequantified by ELISA (FIGS. 7 and 8). As shown in FIG. 6, thetransduction of Arid5A siRNA into RAW 264.7 markedly decreased theexpression level of Arid5A mRNA. As shown in FIG. 7 and FIG. 8, in thecells transduced with Arid5A siRNA, the amount of IL-6 produced inresponse to LPS stimulation was decreased whereas the amount of TNF-αremained unchanged.

Example 4: A Method for Screening for a Candidate Substance Useful forthe Treatment of a Lung Disease

Peritoneal macrophages from C57BL/6 mice (aged 6 to 8 weeks) werestimulated with LPS (1 g/mL) in the presence or absence of a test agent(20 μM). The relative expression levels of mRNAs of Arid5A, IL-6, andTNF-α were determined in the stimulated peritoneal macrophages byquantitative PCR (qPCR). The expression level of Arid5A mRNA wasdetermined 2 hours after LPS stimulation. The expression levels of IL-6mRNA and TNF-α mRNA were determined 2 hours, 6 hours, 12 hours, and 24hours after LPS stimulation. FIGS. 9 to 11 show the results whenchlorpromazine (CPZ) was used as a test agent.

As shown in FIG. 9, the expression of Arid5A mRNA was markedlysuppressed in the peritoneal macrophage stimulated with LPS in thepresence of CPZ 2 hours after LPS stimulation as compared to the absenceof CPZ. Also, as shown in FIG. 10, the expression of IL-6 mRNA wasmarkedly suppressed in the peritoneal macrophage stimulated with LPS inthe presence of CPZ 2 hours and 6 hours after LPS stimulation ascompared to the absence of CPZ. In contrast, as shown in FIG. 11, CPZhad no effect on the expression of TNF-α mRNA.

The results revealed that CPZ was a substance that inhibits theexpression itself of Arid5A mRNA. CPZ was also revealed to be asubstance that inhibits the expression of IL-6 mRNA affected by Arid5Aand has no effect on TNF-α mRNA known not to be affected by Arid5A. Thissuggests that CPZ is a substance that specifically inhibitsstabilization of IL-6 mRNA and is effective in the treatment of lungdiseases involved in inflammatory cytokines such as IL-6. Thus, it isexpected that substances that are effective in the treatment of lungdiseases can be obtained by screening for a substance that inhibits theexpression of Arid5A mRNA within cells, and the substances that inhibitArid5A action can be efficiently screened by screening for a substancethat inhibits the expression of IL-6 mRNA and has no effect on TNF-αmRNA. These methods can be used to efficiently screen for a substanceeffective in the treatment of lung diseases.

INDUSTRIAL APPLICABILITY

The present invention provides pharmaceutical compositions having hightherapeutic and/or prophylactic efficacy for a lung disease. The presentinvention also provides a method for screening for a therapeutic and/orprophylactic agent for a lung disease having high therapeutic efficacy.

1.-4. (canceled)
 5. A method for screening for a candidate substanceuseful for the treatment of a lung disease, wherein the methodcomprises: (a) detecting an effect of test agents on the expressionArid5A and (b) selecting the agents that decrease the expression ofArid5A as compared to the absence of the test agents.
 6. A method forscreening for a candidate substance useful for the treatment of a lungdisease, wherein the method comprises: (a) administering test agents toexperimental animals, (b) determining an effect on expression of Arid5Aby PCR, and (c) selecting the agents that decrease the expression ofArid5A as compared to no administration of the test agents to theexperimental animals.
 7. A method for screening for a candidatesubstance useful for treatment of a lung disease, wherein the methodcomprises: (a) detecting an effect of test agents on function of Arid5Aand (b) selecting the agents that decrease the function of Arid5A ascompared to the absence of the test agents.
 8. The method for screeningfor a candidate substance useful for treatment of a lung diseaseaccording to claim 7, wherein the function of Arid5A is to stabilizeIL-6 mRNA.
 9. The method for screening according to claim 5, wherein thelung disease is pulmonary fibrosis.
 10. (canceled)
 11. The method forscreening according to claim 6, wherein the lung disease is pulmonaryfibrosis.
 12. The method for screening according to claim 7, wherein thelung disease is pulmonary fibrosis.
 13. The method for screeningaccording to claim 8, wherein the lung disease is pulmonary fibrosis.14. The method for screening according to claim 5, wherein the lungdisease is an inflammatory lung disease.
 15. The method for screeningaccording to claim 6, wherein the lung disease is an inflammatory lungdisease.
 16. The method for screening according to claim 7, wherein thelung disease is an inflammatory lung disease.
 17. The method forscreening according to claim 8, wherein the lung disease is aninflammatory lung disease.